6.0 Project Abstract: Epidemiologic research has shown that infection with human papillomavirus (HPV) is a cause of cervical cancer, however not all HPV infections lead to cervical cancer or dysplasia. Early events in an HPV infection are critical to the establishment of an HPV infection that evades the immune system, persists over time, and fuels cancer development and progression. Identification of early molecular events associated with HPV infection and carcinogenesis is a critical step in the development of screening guidelines that distinguish high- risk patients as well as identifying high-value targets for interventions aimed at preventing cancer progression. Among the molecular alterations that occur during tumorigenesis in general, and HPV infection in particular, are aberrant changes in the DNA methylation patterns in both viral and host genomes. Such epigenetic changes are often early events in the cancer process and are responsible, in part, for transcriptional repression of tumor suppressor genes (TSGs) during tumorigenesis. This "epigenetic silencing" is widely viewed as a principal molecular mechanism that drives cancer formation and progression. We hypothesize that aberrant epigenetic events can be exploited to define novel biomarkers with predictive value to improve prognostic and diagnostic information for cervical cancer patients. Limitations of previous studies include unreliable and qualitative methods and a lack of a biologic justification for gene selected in relation to cervical cancer. We intend to overcome these limitations by combining state of the art quantitative DNA methylation analysis of a panel of relevant TSGs with the study of invasive squamous cell cervical carcinoma (SCC) and normal tissues from 50 matched pairs of women for whom we have over 10 years of follow-up data. Specifically, we Aim to 1) quantify the methylation status of a panel of host genes and HPV genomic loci in our set of matched tumor/normal tissues by using pyroseqencing technology and 2) establish quantitative DNA methylation biomarkers useful in the diagnosis and prognosis of cervical cancer. We will quantitatively measure DNA methylation in a panel of 18 host TSGs and 2 regions of the HPV genome in both 50 SCC and 50 control samples using pyrosequencing. We will combine the quantitative DNA methylation information obtained on 50 matched pairs of SCC and normal tissues with pathology and patient information to determine those gene(s) whose aberrant methylation has the highest sensitivity and specificity for the diagnosis of SCC. Furthermore, since we have over 10 years of follow-up data on these patients, we will determine those gene(s) whose aberrant methylation has the highest predictive value for disease free survival. With this strong foundation, we will be placed in a competitive position for R01 funding focused on testing a larger and more comprehensive set of patient samples necessary to firmly establish and validate cervical cancer biomarkers. PUBLIC HEALTH RELEVANCE: Cervical cancer is caused by human papillomavirus and current prevention strategies through screening or vaccination have limitations. Older women or those currently infected are still at risk for cancer. This research will use a resource of specimens that would otherwise be discarded to study differences in epigenetic profiles. The overall goal is to identify a panel of genes that have prognostic and/or diagnostic value in the treatment / management of cervical cancer. Such biomarkers are sorely needed and it is expected that this study will prove an essential advance in this disease.